Biosynthetic studies of thiosugar natural products ; Mechanistic studies of hyoscyamine 6 [beta]-hydroxylase

Natural products produced by microorganisms and plants provide a rich source of novel and diverse chemical entities. Understanding the biosynthetic pathways of these natural products and the chemical mechanisms of the enzymes involved has been essential for modern drug discovery and development. This dissertation describes studies on the biosynthetic pathways of natural products with atypical chemical motifs. Part I describes biosynthetic studies of the lincosamides and albomycins, which are two groups of thiosugar-containing natural products. Lincosamides such as lincomycin A and celesticetin share a thioglycoside core linked to a proline residue but contain different substituents on the sulfur center at C1. It will be demonstrated that three stand-alone nonribosomal peptide synthetase domains are responsible for conjugation between the proline residue and the thiosugar core. In addition, the pyridoxal 5′- phosphate-dependent enzymes LmbF and CcbF are shown to be key enzymes that lead to bifurcation of the lincosamide biosynthetic pathway and differential modification of lincomycin A versus celesticetin. Albomycins are 4′-thiofuranose-containing nucleosides that are conjugated with a ferrichrome-type siderophore through a serine linker residue. It will be shown that AbmM catalyzes formation of the sulfur-bridged furanose core via a radical-mediated mechanism. In addition, it will also be demonstrated that the resulting 4′-thiofuranosyl nucleotide is matured into SB-217452, which is the active antibiotic component of albomycins, via reactions catalyzed by AbmG, AbmH, AbmD, AbmK, AbmF, and AbmJ. Part II describes mechanistic studies of hyoscyamine 6β-hydroxylase (H6H). H6H catalyzes hydroxylation and subsequent oxidative cyclization to form an epoxide ring from an ethylene group during scopolamine alkaloid biosynthesis. Studies using substrate analogues indicate that substrate conformation correlates with selectivity between the dihydroxylation and epoxidation activities of H6H. Based on kinetic analysis, it will also be shown that the presence of a hydroxyl group in the substrate significantly affects the regioselectivity of H atom abstraction during H6H-catalyzed oxidation reactions.